Structural basis of bacteriophage T7 assembly and DNA packaging

噬菌体 T7 组装和 DNA 包装的结构基础

• 批准号: 7339289
• 负责人: Wen Jiang
• 金额: $ 35.13万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-15 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7339289
• 关键词: 3-Dimensional; Algorithms; Antibodies; Antiviral Agents; Arts; Bacteriophage T7; Bacteriophages; Biochemical Genetics; Biological Models; Biology; Capsid; Capsid Proteins; Classification; Complex; Computer software; Computers; Cryoelectron Microscopy; DNA; DNA Packaging; Development; Electron Microscope; Encapsulated; Escherichia coli; Family; Foundations; Genome; Goals; Health; Helix (Snails); Herpesviridae; Human; Imagery; Length; Life Cycle Stages; Methods; Metrizamide; Morphogenesis; Pathway interactions; Personal Satisfaction; Podoviridae; Process; Proteins; Research Personnel; Resolution; Role; Scaffolding Protein; Solutions; Structure; System; T-Phages; Tail; Techniques; Thick; Vaccines; Virion; Virus; Virus Diseases; X-Ray Crystallography; base; density; ear helix; image processing; particle; programs; protein folding; receptor; reconstruction; virus host interaction

DESCRIPTION (provided by applicant): T7 is one of the most studied bacteriophage systems for which a wealth of genetic and biochemical information is known. Similar to other tailed dsDNA phages, the morphogenesis pathway of T7 starts with a DNA-free empty procapsid that is formed through scaffolding protein-assisted assembly. The procapsid undergoes large-scale conformational changes and expands into the capsid II state during the maturation process, which is triggered by DNA packaging through the portal vertex. An infectious particle is formed only after the completion of DNA packaging and tail assembly on the portal vertex. Although phage morphogenesis has been extensively studied for numerous phage systems, including T7, many questions remain to be answered to understand its mechanism. Having been able to obtain some preliminary results on the structures of the T7 capsids, we propose to carry out more systematic studies to elucidate the structural basis of T7 assembly, maturation and DNA packaging. We aim to solve the structures of the capsid shell, the scaffolding proteins, the portal complex, the internal cylindrical core, the tail and the DNA packing. These structures will be examined at each of their capsid states in the morphogenesis pathway to probe their roles in the maturation process. State-of-the-art cryo-electron microscopes and computer 3-D reconstruction techniques will be used to accomplish these tasks. We will use the popular image processing software EMAN, of which the PI is a co-developer, to determine the capsid shell structures to 7-8 A or better resolution by imposing icosahedral symmetry. We will also further develop the image processing algorithms to push for 4-5 A resolution structures for the infectious phage particles. Our recent extension of EMAN to allow the solution of structures of symmetry matches will be used to determine the structures of the scaffolding proteins, portal complex, cylindrical core and tail in the intact capsids at 15 A resolution or better. These proteins do not have icosahedral symmetry and cannot be resolved using classic reconstruction methods that rely on icosahedral averaging. This method will be further developed to allow direct tracing of the dsDNA strands and visualization of dsDNA packing. The T7 structures will form the foundation for understanding the basic processes of assembly, maturation and genome packaging of not only the tailed dsDNA phages, but also the related human herpes viruses.

描述（由申请人提供）：T7是研究最多的噬菌体系统之一，已知其丰富的遗传和生化信息。与其他有尾dsDNA双链体类似，T7的形态发生途径始于通过支架蛋白辅助组装形成的无DNA空原衣壳。原衣壳经历大规模的构象变化，并在成熟过程中扩展到衣壳II状态，这是由通过门顶的DNA包装触发的。只有在门顶完成DNA包装和尾部组装后，才形成感染性颗粒。虽然噬菌体形态发生已被广泛研究了许多噬菌体系统，包括T7，许多问题仍然有待回答，以了解其机制。已经能够获得一些初步结果的T7衣壳的结构，我们建议进行更系统的研究，以阐明T7组装，成熟和DNA包装的结构基础。我们的目标是解决的结构的衣壳壳，支架蛋白，门户网站复杂，内部圆柱形核心，尾巴和DNA包装。这些结构将在形态发生途径中的每个衣壳状态下进行检查，以探测它们在成熟过程中的作用。国家的最先进的冷冻电子显微镜和计算机三维重建技术将被用来完成这些任务。我们将使用流行的图像处理软件EMAN（PI是其共同开发者），通过施加二十面体对称性来确定7-8 A或更好分辨率的衣壳壳结构。我们还将进一步开发图像处理算法，以推动感染性噬菌体颗粒的4-5 A分辨率结构。我们最近对EMAN进行了扩展，以允许解决对称匹配的结构，将用于以15 A或更高的分辨率确定完整衣壳中的支架蛋白、门户复合物、圆柱形核心和尾部的结构。这些蛋白质不具有二十面体对称性，并且不能使用依赖于二十面体平均的经典重建方法来解析。这种方法将进一步发展，以允许直接跟踪的dsDNA链和可视化的dsDNA包装。T7结构将为理解不仅是有尾dsDNA病毒，而且是相关的人类疱疹病毒的组装、成熟和基因组包装的基本过程奠定基础。